INTRODUCTION:

Fungi are harmless commensals found abundantly in the mouth of healthy ‎individuals. They can cause oral mucosal diseases, particularly in ‎immunocompromised individuals^1,2. Candida species constitutes the largest percentage of the normal commensals of the oral cavity in healthy individuals³. Several studies have assessed the commensal status of this fungal genus and found that about 54–71.4% of healthy individuals may carry such yeasts without any symptoms^4-7.

The carriage rate of Candida species in the human body can be influenced by oral disorders such as gingivitis, dental caries, and periodontitis; systemic diseases such as diabetes mellitus; and severe immunosuppression Manfredi^8-10. The growth of Candida is also found among denture wearers who are more likely to develop oral candidiasis⁸.

Studies on Candida characterization among different populations worldwide yielded variable results. However, Candida ‎albicans was found to be the most prevalent yeast species, followed by Candida glabrata, Candida ‎tropicalis, Candida krusei, Candida parapsilosis, and Candida guilliermondii^3,11.

Several lab methods of identification of Candida species which depends on morphological and physiological characteristics of the Candida are used. Likewise, more accurate methods of identification such as T2Candida (PCR with magnetic resonance), PNA-FISH, next-generation sequencing were reported¹².

Although several other fungi can cause oral ‎lesions, Candida albicans is the primary causative agent for oral ‎candidiasis, which has a variety of clinical presentations¹³. Since the oral microbiota can be affected by different environmental and genetic factors, the differential count of Candida species and their response to various antifungal drugs may vary¹³. Thus, Candida glabrata has recently become the second highest cause of candidemia in the United States and Northern Europe¹⁰. Likewise, Candida dubliniensis which are phenotypically identical to Candida albicans is primarily associated with oral candidiasis of human immunodeficiency virus (HIV)-infected individuals^13,14, and a high prevalence of ‎Candida dubliniensis was found in diabetic patients, which might be misdiagnosed with its ‎morphologically related species, Candida albicans^15,16,17. Several studies reported that Candida is significantly higher ‎among people with diabetes ‎than ‎non-diabetics^4,114,18,19.

The changing spectrum of Candida species in the oral environment and their antifungal susceptibility pattern, especially among the immunocompromised individuals, have made the identification of species level and the detection of drug resistance significant for quality patient care²⁰. The aims of this study are to identify and specify oral fungal species among dental patients and to assess their susceptibility to the commonly used antifungal drugs.

MATERIALS AND METHODS:

Demography and study design:

Oral rinses were collected from 120 consecutive patients who visited 3 different dental clinics in Benghazi city during the year 2021. The patients included in this study are adults, aged 18 years or above, not receiving chemotherapy or radiotherapy, and not taking antifungal drugs or antibiotics. The samples were taken in full compliance with the measures and directions implicated in dealing with cases during the COVID-19 pandemic. A total of 59 cases (49.1%) had positive fungi growth. This study was conducted in the full conformance with principle of the Helsinki Declaration. The ethical approval was obtained by the research ethics review committee of the college of dentistry, Benghazi University.

Sample Collection: Oral rinses were collected by standard methods, and phosphate-buffered saline in oral rinses is used, and the plating of saliva samples is done. Species identification and antifungal susceptibility tests were performed, and then the rinses were immediately transferred to a microbiology laboratory.

Fungus culturing and isolations in the laboratory:

Oral rinses were plated using imprint culture on Sabouraud Dextrose Agar (SDA). The pH was adjusted to approximately 5.6 in order to enhance the growth of fungi. To slightly inhibit the bacterial growth, 1g of cefotaxime (455.47g/mol) was dissolved in sterile water and added to 1L of SDA medium, then poured aseptically into Petri dishes and incubated at 36±1°C for 48 h (Figure 1).

Figure 1: Candida spp. plates showing the growth and purity of the colonies.

Gram stain and germ tube testing:

There were 59(49.1%) positive growths of candida out of 120 collected samples, stained with gram stain and further subjected to species identification and antifungal susceptibility using a semi-automated microbial identification system. A thin smear from the positive colonies were prepared on microscope slides, added two drops of lactose phenol cotton blue, covered with a coverslip, and viewed using a light microscope at 40× magnification. Likewise, another smear was prepared for Gram stain and visualized under a light microscope (Figure 2). After successful cultivation, Candida albicans was further identified by germ tube test (Figure 3).

Figure 2: Photomicrographs of smears of Gram stain demonstrating the shape of Candida.

Figure 3: Photomicrographs of smears of Gram stain showing germ tube formations.

Species identifications:

Integral System Yeasts Plus (ISYP) was used for the identification of fungal strains. The species identification steps were carried out according to the purification protocols of the ISYP: (Ref 71822-79822)-Rev.4/25.07.2012 Kit (Liofilchem, Italy).

Presumptive Identification:

Presumptive identification of the fungal strains, based on assimilation reactions, is performed by evaluating the colour change of wells in the plate. The combination of positive and negative reactions of sugars allows the formation of a numerical code. This enables the identification of the yeasts being examined using the table of codes. One well has a chromogenic substrate that allows you to distinguish some yeasts by observing how the colour of the well changes (Figure 4).

Sensitivity to antimycotics:

According to the growth or inhibition of yeasts in media containing antimycotics and a growth indicator in some wells, the sensitivity of Candida strains is assessed. The wells' changing colour from red to orange shows that the yeast being studied is growing slowly and has a moderate sensitivity to the antimycotic concentration present. The colour changes from red to yellow indicates a growth of the yeast under examination and its resistance to the concentration of antimycotics. No colour change indicates no growth of the yeast under examination and its sensitivity to the concentration of antimycotics. The last well growth does not contain antimycotics, but it contains culture medium and indicator, and it works as growth control.

RESULT:

Growth characteristics:

Out of 120 samples, 59 (49.1%) specimens showed positive growth of yeasts on SDA. Finally, after Gram stains, germ tube formation, and phenotypically tested by different chemicals and fermentation sugars, only 11 different culturable fungal species could be identified from the oral cavity.

Frequency of Candida strains:

Out of the 59 positive isolates, Candida albicans was the most commonly isolated species in 39 cases (66%), followed by Candida tropicalis in 8 (13.6%), Candida dubliniensis in 3 (5.1%), Rhodotorula rubra in 2 (3.4%), and 1 case each for Candida krusei, Cryptococcus albidus, Cryptococcus laurentii, Candida catenulata, Candida stellatoidea, Candida famata, and Trichosporon pullulans (Figure 5).

Figure 5: Frequency of Candida strains in the sample.

Culture and colony characteristics:

The type of colony growth found was “few colonies” in 23 cases (39%), “medium growth” in 23 (39%), “heavy growth” in 12 (20.3%), and “single” in only 1 (1.7%). The colony character was “creamy medium” in 30 cases (50.8%), “creamy large” in 15 (25.4%), “creamy small” in 7 (11.9%), and “mixed shape” in 5 (8.5%). Characteristically, in (Rhodotorula rubra it was “white hard large”, and “white hard medium” in Candida laurentii (1 case each) (Table 1).

Table 1: Culture and colony characteristics of the specimens.

Type of growth	Frequency	Percent
Few colonies	23	39.0
Medium growth	23	39.0
Heavy growth	12	20.3
Single	1	1.7
Total	59	100.0
Colony character
Creamy medium	30	50.8
Creamy large	15	25.4
Creamy small	7	11.9
Mixed shape	5	8.5
White hard large (Rhodotorula rubra)	1	1.7
White hard medium (Cryptococcus laurentii)	1	1.7
Total	59	100.0

Antifungal sensitivity:

Antifungal sensitivity testing was performed using the ISYP system on 10 antifungal agents, according to the manufacturer’s protocols (Ref. 71822-79822)-Rev.4/25.07.2012 Kit (Liofilchem) and the CLSI M27-S3 criteria. Most of the cases were sensitive or intermediately sensitive to the tested antifungals, with an overall relatively low resistance rate

The highest sensitivity percentage was recorded to flucytosine (81.4%), followed by amphotericin (76.3%). The highest intermediate sensitivity was recorded to nystatin (69.5%) and the lowest was to flucytosine (15.3%). Generally, the resistance of the isolated yeasts and fungi in this sample were very low to almost all antifungals, with the highest resistance was to clotrimazole (13.6%) and the lowest to ketoconazole (1.7%). The overall resistance rates for the tested antifungals were as follows: nystatin (6.8%), amphotericin B (6.8%), flucytosine (3.4%), econazole (10.2%), ketoconazole (1.7%), clotrimazole (13.6%), miconazole (10.2%), itraconazole (8.5%), voriconazole (8.5%), and fluconazole (11.9%) (Figure 6 & Table 2).

Table 2: Sensitivity of 59 positive cases to different antifungal agents

No.	Agent	Sensitive (%)	Intermediate (%)	Resistant (%)
1	Nystatin	14 (23.70)	41 (69.50)	4 (6.80)
2	Amphotericin	45 (76.30)	10 (16.90)	4 (6.80)
3	Flucytosine	48 (81.40)	9 (15.30)	2 (3.40)
4	Econazole	25 (42.40)	28 (47.50)	6 (10.20)
5	Ketoconazole	40 (67.80)	18 (30.50)	1 (1.70)
6	Clotrimazole	26 (44.10)	25 (42.40)	8 (13.60)
7	Miconazole	29 (49.20)	24 (40.70)	6 (10.20)
8	Itraconazole	30 (50.80)	24 (40.70)	5 (8.50)
9	Voriconazole	31 (52.50)	23 (39.00)	5 (8.50)
10	Fluconazole	32 (54.20)	20 (33.90)	7 (11.90)

Figure 6: Sensitivity of 59 cases to different antifungal agents.

Table 3: Antifungal susceptibility of different strains of oral fungi

Species	Degree	Nystatin %	Flucytosine %	Clotrimazole %	Itraconazole %	Ketoconazole %	Miconazole %	Voriconazole %	Amphotericin %	Econazole %	Fluconazole %
Candida albicans	Intermediate	76.9	10.3	59.0	48.7	33.3	46.2	46.2	15.4	51.3	38.5
	Resistant	2.6	2.6	7.7	2.6	2.6	5.1	5.1	2.6	7.7	10.3
	Sensitive	20.5	87.2	33.3	48.7	64.1	48.7	48.7	82.1	41.0	51.3
Candida tropicalis	Intermediate	87.5	12.5	12.5	25.0	12.5	25.0	25.0	25.0	75.0	12.5
	Resistant	0	0	0	0	0	0	0	0	0	0
	Sensitive	12.5	87.5	87.5	75.0	87.5	75.0	75.0	75.0	25.0	87.5
Rhodotorula rubra	Intermediate	50.0	50.0	50.0	50.0	50.0	50.0	50.0	50.0	0	100.0
	Resistant	50.0	50.0	50.0	50.0	50.0	50.0	50.0	50.0	50.0	0
	Sensitive	0	0	0	0	0	0	0	0	50	0
Candida dubliniensis	Intermediate	66.7	33.3	33.3	33.3	33.3	33.3	33.3	100.0	33.3	33.3
	Resistant	0	0	0	0		0	0	0	0	0
	Sensitive	33.3	66.7	66.7	66.7	66.7	66.7	66.7	100.0	66.7	66.7
Candida krusei	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	0	0	0	0	0	0	0	0	0	0
	Sensitive	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
Cryptococcus albidus	Intermediate	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
	Resistant	0	0	0	0	0			0	0	0
	Sensitive	0	0	0	0	0			0	0	0
Cryptococcus laurentii	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	0	100.0	100.0	100.0	0	100.0	100.0	0	0	100.0
	Sensitive	100.0	0	0	0	100.0	0	0	100.0	100.0	0
Candida catenulata	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	100.0	100.0	100.0	100.0	0	100.0	0	100.0	100.0	100.0
	Sensitive	0	0	0	0	100.0	0	100.0	0	0	0
Candida stellatoidea	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
	Sensitive	0	0	0	0	0	0	0	0	0	0
Candida famata	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	0	100.0	0	0	0	0	0	0	0	0
	Sensitive	100.0	0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
Trichosporon pullulans	Intermediate	0	0	0	0	0	0	0	0	0	0
	Resistant	0	100.0	0	0	0	0	0	0	0	0
	Sensitive	100.0	0	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0

DISCUSSION:

Candida species are considered normal commensal flora in humans; however, they are opportunistic pathogens capable of causing a variety of infections, particularly among the hospitalized and immunocompromised individuals^20,21,22. Fungal infections in humans range from superficial infections to deeply seated systemic mycosis. The unharmful Candida species present in the oral cavity may become virulent, causing mucosal lesions, under certain conditions. Compared with other ‎microbes, the isolation of Candida from intraoral surfaces is not interpreted as a ‎predictive signal for disease, however, accurate and rapid identification of Candida species may assist the treating clinicians with proper choice of antifungal treatment⁹.

Oral isolates can be defined to the species level by both phenotypic and novel molecular methods; for this, it is essential to use easy, standard, cost-effective lab tests^23,24.The colony-forming unit in this study was varied among different species and predominated by white creamy colonies, smooth with focal wrinkles, and the reversum is light-yellow. However, 75% of them are large or medium-sized, which indicates Candida species. Colonies for other fungi show either white hard medium in R. rubra or large hard colonies for Candida laurentii. The colony morphology and colony character can be helpful in species identification²⁵.

Several studies have documented increased rates of Candida glabrata, Candida dubliniensis, Candida parapsilosis, Candida guilliermondii, Candida tropicalis and Candida krusei in local and systemic fungal infections^19,26,27. Some of these species are resistant to commonly used antifungal agents, hence the need for a timely and accurate method of identification is paramount^27-29. Candida albicans in this study was the predominant species in the isolates, comparable to most previous studies. This study didn’t include patients ‎infected with HIV where oral candidiasis is considered a constant finding. The frequency of Candida dubliniensis in this study was higher than that in other populations as it has been found in 5.1% of the isolates in comparison to 0.81% of HIV-infected Brazilian patients³⁰.

‎Candida dubliniensis is a pathogenic yeast closely phylogenetically related to C. albicans and is commonly associated with oral candidiasis in HIV-positive patients; it can cause both superficial and systemic infections^13,31. It was reported that Candida albicans and Candida dubliniensis species are unique in their ability to produce germ tubes and chlamydospores³².

For treating clinician, the identification ability and drug susceptibility testing is very important in order to prescribe the proper antifungal medications, nevertheless, the current version of the ISYP has many disadvantages, such as the difficulty to differentiate between a sample and standard colour. However, there are many advantages to this system, such as speed and time-saving in getting the crucial results, especially in clinical situations. The ISYP, although it provides satisfactory results, is similar to the other diagnostic tests based on carbohydrate assimilation (aerobic metabolism) and fermentation (anaerobic metabolism) reactions and other biochemical tests, which are complex and time-consuming³³. Therefore, polymerase chain reaction (PCR) based diagnostics is rapidly replacing these methodologies in diagnostic laboratories, due to its precision and rapidity³⁴. These molecular techniques are more accurate, sensitive, specific, and relatively simple and have the potential to greatly decrease the time of identification of pathogens that grow slowly or are difficult to grow. Unfortunately, they require technical skills and expensive equipment³⁵.

Most of the Candida species discussed in this study were susceptible to the available antifungal agents, except Candida catenulata and Candida stellatoidea, which were resistant to all antifungal drugs used. Thus, antifungal susceptibility varies greatly according to the fungal species. The accuracy of identification of the fungus and antifungal susceptibility would contribute to the proper management of patients with serious fungal infections^38,39.

In an Ethiopian study, flucytosine demonstrated the greatest antifungal activity against 81.4% of the Candida isolates. Similar to the findings of this study for this agent, the sensitivity to voriconazole surpluses that of flucytosine in that sample. In one study, Candida krusei was 100% resistant to the antifungal drugs, which is in contrast to this study, where Candida krusei was 100% sensitive to all tested antifungal agents²³. This would give clues about the differences in Candida carriage and their antifungal susceptibility in different populations.

In this study, fluconazole showed promising results against Candida albicans, where 51.3% of Candida albicans were sensitive and 38.5% were intermediately sensitive to it. In contrast, 87.5% of Candida tropicalis were sensitive to fluconazole. It appears that Candida stellatoidea and Candida laurentii were the only fluconazole-resistant strains in this sample. The emergence of such strains has long been expected possibly via a nosocomial spread of such resistant strains.

CONCLUSION:

Oral fungi species are predominated by Candida albicans with a variable antifungal susceptibility; however, most of the oral species in this sample are sensitive to the commonly available antifungal drugs. The species identification and drug susceptibility should be checked, especially in severely immunosuppressed individuals. The combination of species identification and antifungal susceptibility testing is very useful, especially from a clinical point of view. Although ISYP can give good results, other methodologies such as biochemical tests and PCR are emerging in many laboratory diagnostics due to their rapidity and precision.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Microbiology lab staff for their kind support.

REFERENCES:

1. Heo SM. Sung RS. Scannapieco FA. Haase EM. Genetic relationships between Candida albicans strains isolated from dental plaque, trachea, and bronchoalveolar lavage fluid from mechanically ventilated intensive care unit patients. Journal of Oral Microbiology. 2011; 3: 1-10. https://doi:10.3402/jom.v3i0.6362.

2. Varshan R, Gopinath P. Characterization and Biofilm Detection among Clinically Important Candida Species. Research Journal of Pharmacy and Technology. 2016; 9(9):1375-1378.

3. Akram Z. Al-Kheraif A. Kellesarian S. Vohra F. Comparison of oral Candida carriage in waterpipe smokers, cigarette smokers, and non-smokers. Journal of Oral Science. 2018;60: 115-120. https://doi:10.2334/josnusd.17-0090.

4. Hauman C. Thompson I, Theunissen F. Wolfaardt P. Oral carriage of Candida in healthy and HIV-seropositive persons. Oral Surgery Oral Medicine Oral Pathology. 1993;76: 570-572. https://doi:10.1016/0030-4220(93)90064-b.

5. Darwazeh A. al-Bashir A. Oral candidal flora in healthy infants. Journal of Oral Pathology and Medicine. 1995; 24: 361-364. http://doi:10.1111/j.1600-0714.1995.tb01200.x.

6. Kindelan S. Yeoman C. Douglas C. Franklin C. A comparison of intraoral Candida carriage in Sjogren's syndrome patients with healthy xerostomic controls. Oral Surgery Oral Medicine Oral Pathology Oral Radiology Endodontic 1998; 85: 162-167. https://doi:10.1016/s1079-2104(98)90420-6.

7. Blignaut E. Pujol C. Lockhart S. Joly S. Soll D. Ca3 fingerprinting of Candida albicans isolates from human immunodeficiency virus-positive and healthy individuals reveals a new clade in South Africa. Journal of Clinical Microbiolgy. 2002; 40: 826-836. https://doi:10.1128/JCM.40.3.826-836.2002.

8. Manfredi M. Characteristics of Candida isolates from patients with diabetes mellitus [Thesis (Ph.D.)]: University of London. 2006. https://doi:10.1111/j.1399-302X.2006.00274.x.

9. Lyon J. da Costa S. Totti V. Munhoz M. Predisposing conditions for Candida spp. carriage in the oral cavity of denture wearers and individuals with natural teeth. Canadian Journal of Microbiology. 2006; 52: 462-467. https://doi:10.1139/w05-148.

10. Aslani N. Abastabar M. Hedayati M. Shokohi T. et al. Molecular identification and antifungal susceptibility testing of Candida species isolated from dental plaques. Journal of Mycology and Medicine. 2018; 28: 433-436. https://doi:10.1016/j.mycmed.2018.05.006.

11. Negroni M. Gonzalez M. Levin B. Cuesta A. Candida carriage in the oral mucosa of a student population: adhesiveness of the strains and predisposing factors. Rev Argentina Microbiology. 2002; 34: 22-8.

12. Khan Z. Ahmad S. Al-Sweih N. Mokaddas E., et al. Changing trends in epidemiology and antifungal susceptibility patterns of six bloodstream Candida species isolates over a 12-year period in Kuwait. PLoS One. 2019; 14: e0216250, 10.1371/journal.pone.0216250. https://doi:10.1371/journal.pone.0216250.

13. Lamont R. Hajishengallis G. Jenkinson H. Oral microbiology and immunology. 2nd ed. Washington, DC: ASM Press;2014.

14. Zomorodian K. Kavoosi F. Pishdad G. Mehriar P. et al. Prevalence of oral Candida colonization in patients with diabetes mellitus. J Mycol Med. 2016; 26: 103-110.

15. Samaranayake L. Essential microbiology for dentistry. 5th ed. Edinburgh: Churchill Livingstone. 2018. Elsevier; pp. 392.

16. Ge Y. He G. Lin T. Lu G. First isolation of Candida dubliniensis from oral cavities of dermatological patients in Nanjing, China. Mycopathologia. 2011; 172: 465-471. http://10.1007/s11046-011-9447-1.

17. Samrithi Yuvaraj, Gheena S, Gopinath P. Comparison of Oral Candidal Flora of Diabetics and Non Diabetics. Research Journal of Pharmacy and Technology. 2016; 9(10):1645-1646.

18. Rosa E. Rached R. Ignacio S. Rosa R. et al. Phenotypic evaluation of the effect of anaerobiosis on some virulence attributes of Candida albicans. Journal of Medical Microbiology. 2008; 57: 1277-12781. https://doi:10.1099/jmm.0.2008/001107-0.

19. Andersen. K. Kristoffersen A. Ingebretsen A. Vikholt et al. Diversity and antifungal susceptibility of Norwegian Candida glabrata clinical isolates. Journal of Oral Microbiology. 2016; 8: 29849. https://doi:10.3402/jom.v8.29849.

20. Orasch C. Marchetti O. Garbino J. Schrenzel J. et al. Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland. Clinical Microbiology and Infection. 2014; 20: 698-705. https://10.1111/1469-0691.12440.

21. Sangamithra V, Ranbir Verma, S. Sengottuvelu, R. Sumathi. Candida Infections of the Genitourinary Tract. Research Journal of Pharmacy and Technology. 2013:6(10): 1111-1115.

22. Pathmashri V.P, Abirami. A Review on Denture Stomatitis. Research Journal of Pharmacy and Technology. 2016; 9(10): 1809-1811.

23. Cárdenes C. Carrillo-Muñoz A. Arias A. Rodríguez-Alvarez C. et al. Comparative evaluation of four commercial tests for presumptive identification of Candida albicans. Journal of Microbiological Methods. 2004; 59: 293-297. https://10.1016/j.mimet.2004.07.009.

24. Zagazig Egypt. Comparison of methods used in identification of Candida albicans. Research Journal of Pharmacy and Technology. 2018; 11(3): 1164-1168.

25. Buschelman B. Jones R. Pfaller M. Koontz F. Colony morphology of Candida spp. as a guide to species identification. Diagnosis of Microbiolgical Infection Diseases. 1993; 35: 89-91. https://10.1016/s0732-8893(99)00051-6.

26. Bhuyan L. Hassan S. Dash K. Panda A. Candida Species Diversity in Oral Cavity of Type 2 Diabetic Patients and their In vitro Antifungal Susceptibility. Contemporary Clinical Dentistry. 2018; 9: S83-S88. https://10.4103/ccd.ccd_70_18.

27. Tamizh Paavai Tha, Gopinath P. Differentiation of Candida dubliniensis on CHROM agar and Pal’s agar. Research Journal of Pharmacy and Technology. 2016; 9(12):2150-2154.

28. Maysoon Khaleefa Abbas, Fatima Rammadan Abdul, Khetam H. Rasool. Immunological and Enzymatic study of Staphylococcus aureus Bacteria and fungi isolated from oral cavity. Research Journal of Pharmacy and Technology. 2022; 15(7):3119-4.

29. Prasanna Guru. E, Gopinath P. Characterization and detection of biofilm among clinical isolates of Candida species by tube method. Research Journal of Pharmacy and Technology. 2016; 9(12): 2109-2112.

30. Ribeiro Ribeiro A. de Alencar Menezes T. de Melo Alves-Junior S. de Menezes S. Oral carriage of Candida species in HIV-infected patients during highly active antiretroviral therapy (HAART) in Belem, Brazil. Oral Surgery Oral Medicine Oral Pathology Oral Radiology. 2015; 120: 29-33. https://10.1016/j.oooo.2015.03.008.

31. Seyoum E. Bitew A. Mihret A. Distribution of Candida albicans and non-albicans Candida species isolated in different clinical samples and their in vitro antifungal suscetibity profile in Ethiopia. BMC Infectious Disease. 2020; 20: 231. https://10.1186/s12879-020-4883-5.

32. Brown DM, Jabra-Rizk M. Falkler W, Jr. Baqui A. Identification of Candida dubliniensis in a study of HIV-seropositive pediatric dental patients. Pediatric Dentistry. 2000; 22: 234-238. https://pubmed.ncbi.nlm.nih.gov/10846737/.

33. Szweda P. Gucwa K. Naumiuk L. Romanowska E. et al. Evaluation of possibilities in identification and susceptibility testing for Candida glabrata clinical isolates with the Integral System Yeast Plus (ISYP). Acta Microbiology Immunology Hung. 2014; 61: 161-172. https://10.1556/AMicr.61.2014.2.6.

34. Liguori G. Di Onofrio V. Lucariello A. Galle F. et al. Oral candidiasis: a comparison between conventional methods and multiplex polymerase chain reaction for species identification. Oral Microbiology Immunology. 2009; 24: 76-78. https://10.1111/j.1399-302X.2008.00447.x.

35. Bicmen C. Doluca M. Gulat S. Gunduz A. Species level identification and antifungal susceptibility of yeasts isolated from various clinical specimens and evaluation of Integral System Yeasts Plus. New Microbiology. 2012; 35: 327-334. https://www.ncbi.nlm.nih.gov/pubmed/22842602.

36. Raghad S. Ibraheem, Basil A. Abbas. Presence of virulence genes of Candida albicans isolated from women with remarks to Antifungal susceptibility. Research Journal of Pharmacy and Technology. 2022; 15(8): 3751-4

37. Luma T. Ahmed. Genotyping and Antifungal Susceptibility of C. albicans Isolated from Infected women. Research Journal of Pharmacy and Technology. 2019; 12(11): 5171-5176.

38. Souza M. Ortiz S. Mello M. Oliveira Fde M. Severo L. Goebel C. Comparison between four usual methods of identification of candida species. Rev Inst Medicine Tropical Sao Paulo. 2015; 57: 281-287. https://10.1590/S0036-46652015000400002.

39. Aoki T. Moro H. Koshio N. et al. [Results of antifungal susceptibility testing of Candida species and trends of antifungal use in Niigata university medical and dental hospital]. Rinsho Byori. 2010; 58: 658-663. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

Received on 11.11.2022 Modified on 14.06.2023

Research J. Pharm. and Tech 2023; 16(11):5316-5322.

DOI: 10.52711/0974-360X.2023.00861